A functional analysis of the partition module of bacteriophage P1, an organism of considerable interest in its own right (1), is the major topic of our current research. A plasmid partition module is a set of genes, generally two structural genes and an adjacent cis-acting site, that ensures the distribution of plasmid copies to daughter cells such that no cell is deprived of a copy. Three functions of the P1 partition module are being studied: partitioning itself, which may be considered the bacterial equivalent of mitosis; pairing of the cis-acting partition sites (parS), the equivalent of centromere pairing in eukaryotes; and autoregulation of the partition operon. A seredipitous observation led us to recognize that the P1-encoded ParB protein, which binds to parS, can spread outward for several kilobases along the DNA flanking parS, silencing genes in its path (2). We find that the spreading of the P1 partition protein ParB along DNA that flanks its specific binding site appears to require more than simply the tethering of the protein to a DNA site. This conclusion is based on the behavior of ParB-GAL4 fusion proteins of which the two moieties retain their capacities to bind to their specific sites on DNA. The conclusion bears upon the relative merits of two hypotheses (3) to explain gene silencing: formation of a nucleoprotein filament or sequestration at cell membrane sites ("transertion"). Currently we are seeking host mutants that affect ParB-mediated silencing in the hope of understanding the process and perhaps clarifying its relation to partitioning. We are also seeking by other routes the step in partitioning (if any) that may require (or be assisted by) ParB spreading. A priori, replicon pairing at partition sites, long believed to be an essential early step in the prokaryotic partitioning process as it is in mitosis, seemed a likely candidate. However, pairing of parS sites had never been demonstrated in vivo or in vitro. We have now obtained evidence for pairing of parS sites by ParB, using a method which detects the loss of the capacity of DNA supercoils to diffuse across sites as a consequence of their having paired in vivo. The pairing appears not to depend upon extensive ParB spreading and therefore is unlikely to be the step in partitioning that is vulnerable to a blockage of spreading. Our studies of the third aspect of par operon function, its autoregulation, were intitiated as a result of a serendipitous observation made during exploratory experiments on the cellular localization of partition proteins tagged with green fluorescent protein (GFP) and provided from a plasmid source. We observed that a single parS locus, inserted in a chromosomal site, in addition to recruiting ParB-GFP to discrete foci, markedly reduced the background fluorescence in the cytoplasm. Whereas it had long been known that regulation of the par operon is mediated by ParA as repressor and ParB as corepressor, a role for parS had not been noted. We have obtained evidence that ParB spreading contributes significantly to the repression increase that is mediated by parS. If, as it appears, the role of parS in repression is catalytic, then it represents a novel regulatory role for a DNA site. References: 1. M. Lobocka, D. Rose, M. Rusin, A. Samojedny, M. Yarmolinsky and F.C. Blattner, Complete nucleotide sequence of P1, a bacteriophage that lysogenizes as a plasmid: Analysis based on two related isolates, in preparation. 2. O. Rodionov, M. Lobocka and M. Yarmolinsky, Silencing of genes flanking the P1 plasmid centromere, Science, 283: 546-9 (1999). 3. M. Yarmolinsky, Transcriptional silencing, Curr Opin Microbiol 3: 138-143, 2000.